Carotenoids are natural pigments responsible for many of the yellow, orange and red colors seen in living organisms. Carotenoids are 40-carbon (C40) terpenoids generally comprising eight isoprene (C5) units joined together. Linking of the units is reversed at the center of the molecule. “Ketocarotenoid” is a general term for carotenoid pigments that contain a keto group in the ionene ring portion of the molecule, whereas “hydroxycarotenoid” refers to carotenoid pigments that contain a hydroxyl group in the ionene ring. Trivial names and abbreviations will be used throughout this disclosure, with IUPAC-recommended semi-systematic names usually being given in parentheses after first mention of a trivial name.
Carotenoids are synthesized from a five carbon atom metabolic precursor, isopentenyl pyrophosphate (IPP). There are at least two known biosynthetic pathways in the formation of IPP, the universal isoprene unit.
One pathway begins with mevalonic acid, the first specific precursor of terpenoids, formed from acetyl-CoA via HMG-CoA (3-hydroxy-3-methylglutaryl-CoA), that is itself converted to isopentenyl pyrophosphate (IPP). In this pathway, IPP condenses with its isomer dimethylallyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP) that contains 10 carbon atoms. IPP condenses with GPP to produce farnesyl pyrophosphate (FPP) that contains 15 carbon atoms. FPP produces geranylgeranyl pyrophosphate (GGPP) with 20 carbon atoms by condensing with IPP again. Condensation of two GGPP molecules with each other produces colorless phytoene, which is the initial carotenoid.
Studies have also shown the existence of an alternative, mevalonate-independent pathway for IPP formation that was characterized initially in several species of eubacteria, a green alga, and in the plastids of higher plants. The first reaction in this alternative pathway is the transketolase-type condensation reaction of pyruvate and D-glyceraldehylde-3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (DXP) as an intermediate. The intermediate DXP is converted into 2-C-methyl-D-erythritol-4-phosphate that is thereafter converted into IPP. [See Harker et al., FEBS Letters, 448:115–119 (1999).]
Through a series of desaturation reactions, phytoene is converted to phytofluene, ζ-carotene, neurosporene and finally to lycopene. Subsequently, lycopene is converted by a cyclization reaction to β-carotene that contains two β-ionene rings. A keto-group and/or a hydroxyl group are introduced into each ring of β-carotene to thereby synthesize canthaxanthin, zeaxanthin, astaxanthin. [See Britton, Plant Pigments, Goodwin, T. W., ed., London, Academic Press, (1988), pp. 133–182; see also Misawa et al., J. Bacteriol., 177:6575–6584 (1995).]
A hydroxylase enzyme has been shown to convert canthaxanthin to astaxanthin. Similarly, a ketolase enzyme has been shown to convert zeaxanthin to astaxanthin. The ketolase also converts β-carotene to canthaxanthin and the hydroxylase converts β-carotene to zeaxanthin. [See Kajiwara et al., Plant Mol. Biol., 29:343–352 (1995); and Fraser et al., Eur. J. Biochem., 252:229–236 (1998).]
Findings from studies in A. aurantiacum and E. uredovora suggest that the gene(s) that code for the ketolase and hydroxylase are bifunctional in that each of those enzymes can bind and react at one β-ring, and then release the product and rebind, react and release a second product. There are several distinct biosynthesis pathways from β-carotene that can produce astaxanthin using only ketolase and hydroxylase enzymes for each conversion of the intermediary carotenoid. [See Misawa et al., J. Bacteriol., 177:6575–6584 (1995).]
Canthaxanthin, whose structural formula is shown below, is a red xanthophyll carotenoid
that normally does not occur in flower petals and is found in some mushrooms and in the feathers of flamingos. Canthaxanthin is used as a food coloring. It is also used as an oral suntan agent.
Astaxanthin, a red xanthophyll whose structural formula is shown below, is widely used as a pigmenting agent for cultured fishes and shellfishes. The complete biomedical properties of
astaxanthin remain to be elucidated, but initial results suggest that it could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system. [See Tanaka et al., Carcinogenesis 15(1):15–19 (1994), Jyonouchi et al., Nutrition and Cancer 19(3): 269–280 (1993) and Jyonouchi et al., Nutrition and Cancer 16(2): 93–105 (1991).]
Astaxanthin is a carotenoid that occurs particularly in a wide variety of marine animals including fish such as salmonids and sea bream, and crustaceans such as crab, lobster, and shrimp. Because animals generally cannot biosynthesize carotenoids, they obtain those carotenoids present in microorganisms or plants upon which they feed. For this reason, astaxanthin has been widely used as a feed additive for the purpose of red color enhancement for cultured fish and shellfish such as sea bream, salmon, and shrimp and the like. Moreover, astaxanthin is attracting attention as an antioxidant to remove activated oxygen generated in a body, which is causative of a cancer. [See Matuno et al., KAGAKU TO SEIBUTU (Chemistry and Organisms), 28:219–227 (1990).]
Astaxanthin supplied from biological sources, such as crustaceans, yeast, and green alga is limited by low yield and costly extraction methods when compared with that obtained by organic synthetic methods. Usual synthetic methods, however, produce by-products that can be considered unacceptable. It is therefore desirable to find a relatively inexpensive source of (3S, 3′S)-astaxanthin to be used as a feed supplement in aquaculture and as a valuable chemical for other industrial uses.
Astaxanthin has been found to have diverse biological functions. It is a vitamin A precursor, acts as a scavenger and/or quencher of free radicals and active oxygen species, is seemingly a preventative against cancer and has been shown to enhance the immune response. [See Misawa et al., J. Bacteriol., 177:6575–6584 (1995).] From studies of the properties of astaxanthin, it is a carotenoid of great interest to the pharmaceutical, “nutraceutical” (as a pre-cursor to vitamin A and other properties), and food industries.
Sources of astaxanthin include crustaceans such as a krill in the Antarctic Ocean, cultured products of the yeast Phaffia, cultured products of a green alga Haematococcus pluvialis, and products obtained by organic synthetic methods. However, when crustaceans such as a krill or the like are used, a great deal of work and expense are required for the isolation of astaxanthin from contaminants such as lipids and the like during the harvesting and extraction. Moreover, in the case of the cultured product of the yeast Phaffia, a great deal of expense is required for the gathering and extraction of astaxanthin because the yeast has rigid cell walls and produces astaxanthin only in a low yield.
Although H. pluvialis may produce one of the highest levels of astaxanthin (0.5–2 percent dry weight) among organisms, most of the astaxanthin synthesized by this alga is esterified. Such esterification may reduce its bioavilability to fish. Furthermore, H. pluvialis needs high light levels for astaxanthin formation.
For these reasons, astaxanthin produced from biological sources is deemed to be inferior to that obtained by the organic synthetic methods on the basis of cost. The organic synthetic methods however have a problem of by-products produced during the reactions in consideration of use of astaxanthin as a feed for fish and shellfish, and as an additive to foods. The products obtained by the organic synthetic methods can be contrary to some consumers' preference for naturally produced products. Thus, it would be desirable to supply an inexpensive astaxanthin that is free from contaminating side products and is produced from a biological source.
One approach to increase the productivity of astaxanthin or canthaxanthin production in a biological system is to use genetic engineering technology. Genes suitable for this conversion have been reported.
Ketolase (β-carotene ketolase or β-carotene oxygenase or just ketolase), as used herein, refers to the enzyme that causes a ketone (oxo) group to be added to the 4-position carbon atom of a carotenoid β-ionene ring to form various ketocarotenoid compounds in the later stages of the carotenoid biosynthesis pathway. There are several sources of genes that encode a ketolase enzyme that can convert carotenoid a β-ionene ring into a 4-keto-β-ionene ring that is present in canthaxanthin and astaxanthin.
For example, Misawa et al. (See, U.S. Pat. No. 6,150,130) specified DNA sequences including one isolated from the marine bacteria Agrobacterium aurantiacum sp. nov. MK1 or Alcaligenes sp. PC-1 that encodes a gene, referred to as crtW, used in the production of astaxanthin from a carotenoid β-ionene ring compound as a substrate by way of 4-ketozeaxanthin. Cunningham (See, WO 99/61652) reported isolation of a DNA that encodes a protein having ketolase enzyme activity from Adonis aestivalis, a plant species having deep red flower color due in part to the accumulation of the ketocarotenoid astaxanthin.
Two different genes that can convert a carotenoid β-ionene ring compound into astaxanthin have been isolated from the green alga Haematococcus pluvialis. The cloned cDNAs were shown to encode different β-carotene ketolase enzymes that convert a β-ionene ring methylene group to a β-ionene ring keto group (thus acting as a “ketolase”).
One gene product has been designated as bkt by the first group to report its isolation encodes a polypeptide having a beta-C-4-oxygenase activity for the production of (3S,3′S)-astaxanthin from a host microorganism or a plant. [See Kajiwara et al., Plant Molecular Biology, 29:343–352 (1995); and Kajiwara et al. U.S. Pat. No. 5,910,433.] The second astaxanthin-forming gene and its translation product are referred to as crtO by the researchers that isolated the gene that encodes an enzyme that synthesizes astaxanthin. [See Harker et al., FEBS Letters, 404:129–134 (1997) and Lotan et al., FEBS Letters, 364:125–128 (1995); and Hirschberg et al. U.S. Pat. No. 5,965,795.] The crtO cDNA of the Hirschberg group had sequence identity of approximately 75–76 percent with the bkt gene of the Kajiwara group. The protein product of the crtO gene had a sequence identity of approximately 78 percent of that encoded by the bkt gene. The Lotan et al. paper reported a negative result in trying to transform zeaxanathin expressed in E. coli with the product of the crtO gene of H. pluvialis. 
Genes that encode enzymes that can form astaxanthin from a carotenoid β-ionene ring compound such as zeaxanthin or β-carotene were also found in the marine bacteria Agrobacterium aurantiacum and Alcaligenes PC-1. These genes and their enzyme products, called crtW, exhibit about 75 percent identity to each other and about 37 percent homology to the bkt gene product of the H. pluvialis. The three β-carotene ketolases have four highly conserved regions. [See Kajiwara et al., Plant Mol. Biol., 29:343–352 (1995); and also Misawa et al. U.S. Pat. No. 5,811,273 and No. 5,972,690.]
The term “hydroxylase” as used herein, refers to the gene or encoded enzyme that causes a hydroxyl group to be added to a carbon atom at the 3-position of a carotenoid β-ionene ring to form zeaxanthin or another hydroxylated intermediate in the later stages of the carotenoid biosynthesis pathway. More specifically, a contemplated hydroxylase (or β-carotene hydroxylase) is an enzyme that converts β-carotene or a 4-keto-β-carotene into one or more compounds that are hydroxylated at the 3-positon of the β-ring.
Different sources encoding a hydroxylase enzyme that converts carotenoid a β-ionene ring into a 3-hydroxy-β-ionene ring has been identified. The crtZ gene of Erwinia uredovora encodes one such hydroxylase. [See Kajiwara et al., Plant Mol. Biol., 29:343–352 (1995).] A suitable hydroxylase is also encoded by the crtz gene of Erwinia herbicola. [See Ausich et. al., U.S. Pat. No. 5,684,238 (1997).]
A carotenoid biosynthesis gene cluster was identified in astaxanthin-producing bacteria, Agrobacterium aurantiacum. A crtz gene from that cluster was identified as coding for β-carotene hydroxylase. [See Misawa et al., J. Bacteriol., 177:6575–6584 (1995).] In the Misawa et al. disclosure, A. aurantiacum crtZ gene was introduced to an E. coli transformant that accumulated all-trans-β-carotene. The transformant so formed produced zeaxanthin.
Although the experimental data did not demonstrate the ultimate production of astaxanthin, those data did demonstrate that the Agrobacterium aurantiacum crtZ gene encoded a hydroxylase. Because A. aurantiacum is an astaxanthin-producer, it is inferred that the hydroxylase, demonstrated to be bi-functional, converts canthaxanthin to astaxanthin. At minimum, this hydroxylase converts β-carotene to zeaxanthin, an intermediary in the carotenoid biosynthesis pathway from β-carotene into astaxanthin.
Furthermore, a crtZ gene having about a 90 percent identity to the crtZ gene of A. aurantiacum has been identified in Alcaligenes sp. strain PC-1. The function of the crtZ in A. aurantiacum and in the Erwinia family is as a hydroxylase. [See Misawa, “J. Bacteriol., 177:6575–6584 (1995).]
A carotenoid biosynthesis gene cluster for the production of astaxanthin has been isolated from A. aurantiacum. The five-carotenogenic genes with the same orientation that were found in this cluster, have been designated crtW, crtZ, crtY, crtI, and crtB respectively. The stop codons of the individual crt genes, with the exception of crtB, overlapped with the start codons of the following crt genes. [See Misawa, “J. Bacteriol., 177:6575–6584 (1995).] DNA sequences of A. aurantiacum and Alcaligenes sp. strain PC-1 for the crtW, crtZ and crtY genes that encode a ketolase, hydroxylase and lycopene cyclase enzyme are disclosed in U.S. Pat. No. 5,811,273 and No. 5,972,690.
A gene cluster encoding the enzymes for a carotenoid biosynthesis pathway has been also cloned from the purple photosynthetic bacterium Rhodobacter capsulatus. [See Armstrong et al., Mol. Gen. Genet., 216:254–268 (1989).]
A similar cluster for carotenoid biosynthesis from ubiquitous precursors such as farnesyl pyrophosphate and geranyl pyrophosphate has been cloned from the non-photosynthetic bacteria Erwinia herbicola. The members of the gene cluster identified from E. herbicola include genes referred to as encoding GGPP synthase, phytoene synthase, phytoene dehydrogenase (4H), lycopene cyclase, and β-carotene hydroxylase genes. [See Ausich et al. U.S. Pat. No. 5,684,238; Sandmann et al., FEMS Microbiol. Lett., 71:77–82 (1990); Hundle et al., Photochem. Photobiol., 54:89–93 (1991); and Schnurr et al., FEMS Microbiol. Lett. 78:157–162(1991).]
Yet another carotenoid biosynthesis gene cluster has been cloned from Erwinia uredovora. In E. uredovora, these genes have been identified as crtE, crtB, crtI, and crtZ. [See Misawa et al. U.S. Pat. No. 5,429,939; and Misawa et al., J. Bacteriol., 172:6704–6712 (1990).]
In the Erwinia and Rhodobacter species, crtE encodes GGPP synthase. CrtE, however, is absent in A. aurantiacum. Although the initial substrates of the enzymes encoded in the above gene clusters differ between species, it is the latter crt genes that have been demonstrated to play a significant role in the production of astaxanthin from the carotenoid precursor present. The production of astaxanthin in the marine Agrobacteria suggests that crtW and crtZ gene products, as identified in the various species, are primarily responsible for the conversion of β-carotene to astaxanthin via ketocarotenoid intermediates. [See Misawa et al., J. Bacteriol., 177:6575–6584 (1995).]
The studies reported in Fraser et al., Eur. J. Biochem., 252:229–236 (1998) indicate that β-carotene is the preferred substrate as compared to zeaxanthin for the A. aurantiacum ketolase and other possible oxygenated β-carotene derivatives when studied in an in vitro environment. Those authors also reported a less pronounced preference by the A. aurantiacum hydroxylase enzyme for a compound that contained a hydroxyl group on one ring and a keto group on the other (3-hydroxyechinenone) as compared to β-carotene or other oxygenated β-carotene derivatives. It is unknown and unpredictable as to whether the observed in vitro substrate preferences apply in vivo in A. aurantiacum or in a plant transformed with genes for those enzymes. It is also unknown and unpredictable as to whether enzymes encoded by other organisms behave similarly to that of A. aurantiacum in vitro or in vivo after transformation into the cells of a higher plant.
In many plants, lycopene is a branch point in carotenoid biosynthesis. Thus, some of the plant's lycopene is made into beta-carotene and zeaxanthin, and sometimes zeaxanthin diglucoside, whereas remaining portions of lycopene are formed into alpha-carotene and lutein (3,3′-dihydroxy-α-carotene), another hydroxylated compound.
Carotenoids in higher plants; i.e., angiosperms, are found in plastids; i.e., chloroplasts and chromoplasts. Plastids are intracellular storage bodies that differ from vacuoles in being surrounded by a double membrane rather than a single membrane. Plastids such as chloroplasts can also contain their own DNA and ribosomes, can reproduce independently and synthesize some of their own proteins. Plastids thus share several characteristics of mitochondria.
In leaves, carotenoids are usually present in the grana of chloroplasts where they provide a photoprotective function. Beta-carotene and lutein are the predominant carotenoids, with the epoxidized carotenoids violaxanthin and neoxanthin being present in smaller amounts. Carotenoids accumulate in developing chromoplasts of flower petals, usually with the disappearance of chlorophyll. As in flower petals, carotenoids appear in fruit chromoplasts as they develop from chloroplasts.
In a typical biosynthesis pathway for the production of β-carotene, enzymes convert geranylgeranyl pyrophosphate of the central isoprenoid pathway through phytoene and lycopene to β-carotene. Zeaxanthin, canthaxanthin and astaxanthin are among the xanthophylls that arise from β-carotene. Most enzymes that take part in conversion of phytoene to carotenes and xanthophylls are labile, membrane-associated proteins that lose activity upon solubilization. [See Breyer et al., Eur. J. Biochem., 153:341–346(1985); see also Hirschberg et al. U.S. Pat. No. 5,965,795 (1999)].
At the present time only a few plants are widely used for commercial colored carotenoid production. However, the productivity of colored carotenoid synthesis in most of these plants is relatively low and the resulting carotenoids are expensively produced. In addition, canthaxanthin and astaxanthin are not carotenoids that are so produced.
Hirschberg et al. U.S. Pat. No. 5,965,795 teaches that astaxanthin could be produced in the nectaries of transgenic tobacco plants. Those transgenic plants were prepared by Argobacterium tumifaciens-mediated transformation of tobacco plants using a vector that contained a ketolase-encoding gene from H. pluvialis denominated crtO along with the Pds gene from tomato as the promoter and to encode a leader sequence. The Pds gene was said by those workers to direct transcription and expression in chloroplasts and/or chromoplast-containing tissues of plants.
Results from that transformation indicated the production of five ketone-containing carotenoids, including astaxanthin in the nectary. Those results indicated that about 75 percent of the carotenoids found in the flower of the transformed plant contained a keto group. However, no evidence was presented as to the quantity of initial carotenoid present in the flower, nor about the about the amount of total carotenoid actually produced, nor about the production of carotenoids in the petals or reproductive parts of the flower.
The Tagetes genus is a member of the plant family Compositae, alternatively known as Asteraceae, and comprises some thirty species of strongly scented annual or perennial herbs. Tagetes are native from Arizona and New Mexico to Argentina. [See Hortus Third A Concise Dictionary of Plants Cultivated in the United States and Canada, MacMillan Publishing Company, New York (1976).] Cultivated genera include Tagetes erecta, commonly referred to as African marigold, Tagetes patula, commonly referred to as French marigold, Tagetes erecta x patula, commonly referred to as Triploid marigolds, and Tagetes tenuifolia, also known as Tagetes signata or signet marigold.
A marigold inflorescence is a solitary head comprised of a dense cluster of several hundred sessile or subsessile small flowers also known as florets. Marigolds have radiate flower heads with outer ray florets that are ligulate or strap-shaped around the central tubular-shaped disk florets. Some forms of marigold flower heads have most of their disk flowers transformed into ray flowers and contain few, if any, disk flowers. Such flower heads are referred to as double-flowered.
The ray flowers or florets are often referred to as petals by lay persons who may also refer to the flower heads as flowers. For ease of understanding, marigold flower heads will be referred to herein as flowers or flower heads, whereas the flower head-component flowers or florets, stamens, and pistils will be referred to as petals.
Cultivated marigolds possess showy flowers and are useful for ornamental purposes. In addition, the genus is recognized as a source for natural color, essential oils, and thiophenes. Dried marigold petals and marigold petal concentrates obtained from so-called xanthophyll marigolds are used as feed additives in the poultry industry to intensify the yellow color of egg yolks and broiler skin. [See Piccalia et al., Ind. Crops and Prod., 8:45–51 (1998).] The carotenoids desired in poultry tissues are a function of their dietary concentration, because poultry do not have the ability to synthesize carotenoids de novo. [See Balnave et al., Asian-Australiasian J. Animal Sci., 9(5): 515–517 (1996).]
The pigmenting ability of marigold petal meal resides largely in the carotenoid fraction known as the xanthophylls, primarily lutein esters. [See Piccalia et al., Ind. Crops and Prod., 8:45–51 (1998)]. The xanthophyll zeaxanthin, also found in marigold petals, has been shown to be effective as a broiler pigmenter, producing a highly acceptable yellow to yellow-orange color [See Marusich et al., Poultry Sci., 55:1486–1494 (1976)]. Of the xanthophylls, the pigments lutein and zeaxanthin are the most abundant in commercially available hybrids. Structural formulas for lutein and zeaxanthin are shown below.

Each lutein and zeaxanthin terminal ring structure contains one hydroxyl group, so that each molecule contains two hydroxyl groups. Lutein is believed to be biologically produced by two separate hydroxylations of α-carotene, whereas zeaxanthin is believed to be biologically produced by two separate hydroxylations of β-carotene. Both α-carotene and β-carotene are understood to be formed by the action of appropriate cyclase enzymes on δ-carotene and γ-carotene, respectively, which are formed by cyclization of lycopene. Lycopene, δ-carotene, γ-carotene, (α-carotene and β-carotene are each hydrocarbon carotenoids, and with their 40-carbon precursors are referred to as carotenes. Oxygenated carotenoids such as lutein, zeaxanthin, astaxanthin and violaxanthin are referred to as xanthophylls.
FIG. 1 shows a schematic representation of the biological synthesis pathway for the production of lutein and zeaxanthin and later products from phytoene, the first C40 carotenoid in the pathway. Lutein and zeaxanthin are present in marigold petals primarily as mono- and dβ-esters of fatty acids. FIG. 1 also notes epoxide-containing later products that can arise from zeaxanthin, of which violaxanthin is an intermediate in the abscisic acid synthetic pathway.
Carotenoids have been found in various higher plants in storage organs and in flower petals. For example, marigold flower petals accumulate large quantities of esterified lutein as their predominant xanthophyll carotenoid (about 75 to more than 90 percent), with smaller amounts of esterified zeaxanthin. Besides lutein and zeaxanthin, marigold flower petals also typically exhibit a small accumulation of β-carotene and epoxidized xanthophylls, but do not produce or accumulate canthaxanthin or astaxanthin because a 4-keto-β-ionene ring-forming enzyme is absent in naturally-occurring marigolds or their hybrids.
Xanthophyll marigolds differ in several characteristics from ornamental marigolds. First and foremost, xanthophyll marigolds are used as an extractable source for carotenoids and have plant habits that differ from ornamental marigolds. Ornamental marigolds typically grow only about 45 to about 60 cm from the ground, whereas xanthophyll marigolds grow to about 65 to about 70 cm from the ground. Xanthophyll marigolds grow in a bushier habit than do ornamental marigolds, and can be grown as row crops whereas ornamental marigolds typically cannot. Xanthophyll marigolds are typically dark orange in color, whereas ornamentals can be white, yellow, or orange in color, or can have mixed colors, including mahogany colors due to anthocyanin pigments that are less abundant in xanthophyll marigolds.
One way to increase the productive capacity of biosynthesis is to apply recombinant DNA technology. Thus, it would be desirable to produce colored carotenoids generally and, with the use of recent advances in determining carotenoid biosynthesis from β-carotene to canthaxanthin or astaxanthin, or both, to control the production of carotenoids, specifically canthaxanthin and astaxanthin. That type of production permits control over quality, quantity and selection of the most suitable and efficient producer organisms. The latter is especially important for commercial production economics and therefore availability to consumers.
It would be advantageous if a marigold or other plants were available whose flowers produced large amounts of β-carotene, canthaxanthin, zeaxanthin, or other astaxanthin precursors and small amounts or no lutein so that such plants could be transformed with one or more of an appropriate hydroxylase gene and an appropriate ketolase gene to produce astaxanthin from the flowers of the resulting transformants. The invention discussed hereinafter relates in some embodiments to such transformed plants, and particularly to transformed marigold plants.